ANTIBACTERIAL PEPTIDE

PEPTIDE ANITBACTERIEN

English Abstract

ANTIBACTERIAL PEPTIDE
Abstract of the Disclosure
It has been found that dipeptides containing a
3-halo-D-alanine C-terminus are powerful antibacterials
and produce a highly useful synergistic effect with anti-
biotics.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for preparing a peptide of the formula:
<IMG>
wherein the shown amino acid is in the D-configuration, X
is a halogen selected from the group consisting of: chlorine
and fluorine and R is the acyl moiety of an .alpha.-aminoacid in
the L-configuration; said process comprising: coupling an
alanine selected from the group consisiting of: .beta.-chloro-
and .beta.-fluoro-D-alanine with a reactant selected from the
group consisting of: an active ester of an N,.alpha.-protected
glycine, an amino acid in the L-configuration and an N,.alpha.-
alkyl homolog thereof.
2. A process as defined in claim 1, wherein the
.alpha.-amino group of said acyl moiety carries an additional
moiety selected from the group consisting of: a fatty acid
acyl group, an aminoloweralkyl acyl group, a loweralkyl group
and a corresponding loweralkyl ester of the said peptide.
3. A process as defined in claim 2, wherein any N,.alpha.-
amino group of said L-configuration amino acid carries a
protecting group; and removing said protecting group.
4. A process as defined in claim 3, wherein the
carhoxy group of said alanine is esterified with a loweralkyl
alcohol.
5. A process as defined in claim 1, wherein X is
fluorine and R is the acyl moiety of L-serine.
6. A process as defined in claim 1, wherein said
peptide has the formula:
<IMG>
14

wherein R' is selected from the group consisting of: hydrogen
and an alkyl group of 1 to 8 carbon atoms.
7. A process as defined in claim 6, wherein X is
fluorine and R' is methyl.
8. A process as defined in claim 6, wherein X is
fluorine and R' is isobutyl.
9. A process as defined in claim 6, wherein X is
fluorine and R' is isopropyl.
10. A process as defined in claim 6, wherein X is
chlorine and R' is methyl.
11. A process as defined in claim 6, wherein X is
chlorine and R' is isobutyl.
12. A peptide of the formula:
<IMG>
wherein the shown amino acid is in the D-configuration, X
is a halogen seleeted from the group consisting of: chlorine
and fluorine and R is the acyl moiety of an .alpha.-aminoacid in
the L-configuration; or a pharmaceutically acceptable acid
addition salt thereof; when prepared by the process defined
in claim 1, or an obvious chemical equivalent thereof.
13. A peptide of the formula:
<IMG>
wherein the shown amino acid is in the D-configuration, X
is a halogen selected from the group consisting of: chlorine
and fluorine and R is the acyl moiety of an .alpha.-aminoacid in
the L-configuration, and wherein the .alpha.-amino group of said acyl
moiety carries an additional moiety selected from the group
consisting of: a fatty acid acyl group, an aminoloweralkyl
acyl group, a loweralkyl group and a corresponding lower-
alkyl ester of the said peptide; or a pharmaceutically

acceptable acid addition salt thereof; when prepared by the
process defined in claim 2, or an obvious chemical equivalent
thereof.
14. A peptide of the formula:
<IMG>
wherein the shown amino acid is in the D-configuration, X
is a halogen selected from the group consisting of: chlorine
and fluorine, R is the acyl moiety of an .alpha.-aminoacid in the
L-configuration and Y is loweralkyl; or a pharmaceutically
acceptable acid addition salt thereof; when prepared by the
process defined in claim 4, or an obvious chemical equivalent
thereof.
15. A peptide of the formula:
<IMG>
wherein the shown amino acid is in the D-configuration and
Rs is the acyl moietv of L-serine; or a pharmaceutically
acceptable acid addition salt thereof; when prepared by the
process defined in claim 5, or an obvious chemical equivalent
thereof.
16. A peptide of the formula:
<IMG>
wherein X is a halogen selected from the group consisting of:
chlorine and fluorine and R' is selected from the group
consisting of: hydrogen and an alkyl group of 1 to 8 carbon
atoms; or a pharmaceutically acceptable acid addition salt
thereof; when prepared by the process defined in claim 6, or
an obvious chemical equivalent thereof.
16

17. A pepticle of the formula:
<IMG>
or a pharmaceutically acceptable acid addition salt thereof;
when prepared by the process defined in claim 7, or an obvious
chemical equivalent thereof.
18. A peptide of the formula:
<IMG>
or a pharmaceutically acceptable acid addition salt thereof;
when prepared by the process defined in claim 8, or an obvious
chemical equivalent thereof.
19. A peptide of the formula:
<IMG>
or a pharmaceutically acceptable acid addition salt thereof;
when prepared by the process defined in claim 9, or an obvious
chemical equivalent thereof.
20. A peptide of the formula:
<IMG>
or a pharmaceutically acceptable acid addition salt thereof;
when prepared by the process defined in claim 10, or an
obvious chemical equivalent thereof.
21. A peptide of the formula:
<IMG>
17

or a pharmaceutically acceptable acid addition salt thereof;
when prepared by the process defined in claim 11, or an
obvious chemical equivalent thereof.
18

Description

--1--
Detailed Description of the Invention
As micro-oryanisms become resistant to known
antibiotics continued effort is needed to find new com-
pounds or combinations of compounds which effectively --
inhibit bacteria growth.
It has now been found that a peptide of the
formula
R-NH-CH-COOH
CH2X
wherein the shown aminoacid is in the D-configuration, X
is chlorine or fluorine, and R is the acyl moiety of an
a-aminoacid in the L-configuration, wherein the a-amino
group may carry a fatty acid acyl group or an aminolower-
alkyl acyl group or a loweralkyl group, or the correspond-
ing loweralkyl esters of said dipeptide, or nontoxic acidaddition salts thereof, are useful antibacterials7 they
also represent powerful synergists for D-cycloserine and
other antibiotics.
The above moiety R particulary represents the
known, protein-derived aminoacids, including glycine which,
of course, does not have a chiral center. The definition
also includes other aminoacids where the amino group is
attached to the 2- or a-poSitiOn of the acid. The amino
group of substituent R may also carry an acyl group of a
lower fatty acid or a loweralkyl group, primarily methyl,
propyl, tert. butyl, acetyl, propionyl isobutyryl and the
like. The protein-derived aminoacid may be represented by
leucine, valine, norvaline, proline, serine, tyrosine,
alanine, phenylalanine, threonine, methionine, glutamine,
histidine, arginine, lysine and tryptophane. The new di-
peptides have the unnatural sequence of an L-aminoacid (or
glycine) coupled to D-haloalanine. Such a L-D sequence is
usually restricted to the cell wall components of micro-
organisms and its antibacterial activity is completely
unexpected.
The new dipeptide can easily be synthesized by
coupling the known ~-fluoro- (or chloro-)-D-alanine with
... ~
.

--2--
an active ester of a N~-protected glycine or an aminoacid
in the L-configuration or a N~-alkyl homolog thereof.
Among the active esters, the hydroxysuccinimide, penta-
chlorophenyl, 4-nitrophenyl, 2,4,5-trichlorophenyl, a
fluorophenyl, N-hydroxyisobornyldicarboximide or similarly
familiar esters of RCOO- can be used for the coupling re-
action. The N ~ group and any other sensitive functional
group in the aminoacid moiety represented by R above can
be protected with the usual well-known groups that can
subsequently be removed by a mild chemical reaction which
does not affect the peptide bond formed. Among the groups
frequently used as temporary protection are the carboben-
zoxy (hereinafter identifed as Z) or the tert. butoxy-
carbonyl for amino groups, particularly the N~- group,
while benzyl or other moieties can be used to protect the
hydroxy groups in serine, tyrosine or hydroxyproline or
the imidazol group of histidine. Hydrogenation will re-
move said benzyl group after the peptide coupling has been
effected and treatment with hydrobromic acid or hydro-
fluoric acid will remove other protective groups used by
the skilled artisan, without cleaving the peptide bond.
The free acid can be converted into the desired alkyl
ester in known fashion and/or the N~- group can be acy-
lated in known manner.
In order to illustrate the preparation of
the new peptides, reference is made to the following
examples which, however, are not intended to limit this
invention in any respect. In all examples, the optical
rotations were taken at 25C. in lN HCl at the concentra-
tions given.
Example 1
a) To a stirred solution of 214.2 mg. of
~-fluoro-D-alanine and 420 mg. of sodium bicarbonate in
S ml. of water was added a solution of 800.8 mg. of
carbobenzoxy-L-alanine-N-hydroxysuccinimide ester in 5
ml. of 1,2-dimethoxyethane. After stirring overnight at
ambient temperature, the solution was concentrated to

113'7~
--3--
a syrup under reduced pressure. The residue was dis-
solved in 10 ml. of water and acidified with lN-hydro-
chloric acid to precipitate 521 mg. of N-carbobenzoxy-
L-alanyl- ~fluoro-D-alanine, m.p. 156-7C.
b) A solution of 2.48 g. of this protected
dipeptide in 10 ml. of 32~ hydrobromic acid in acetic
acid was stirred at room temperature for 30 minutes.
A gummy solid was precipitated by the addition of ether.
This material was washed with 3 portions of ether by de-
cantation and crystallized from wet acetic acid, produc-
ing 1.44 g. of L-alanyl-~-fluoro-D-alanine hydrobromide,
m.p. 203-5C. (with decomposition); [a]D + 25.3 (c, 1.1).
Example 2
a) By repeating the process of Example l(a),
but starting with 320 mg. of ~-chloro-D-alanine hydro-
chloride and 588 mg. of sodium carbonate, 647 mg. of N-
carbobenzoxy-L-alanyl-~-chloro-D-alanine was obtained;
m.p. 168-70C.
b) A solution of 736 mg. of this protected
peptide in 101 ml. of methanol containing one equivalent
of HCl was hydrogenated over 0.15 g. of 5% Pd on carbon.
The catalyst was removed by filtration after the calcu-
lated amount of gaseous hydrogen had been absorbed. The
catalyst was washed with methanol which was combined with
the filtrate. This mixture was evaporated to dryness and
the residue was placed on a 1.5 x 40 cm. column charged
with a strongly basic polystyrene ion exchange resin and
eluted with 0.1 molar ammonium acetate buffer of pH 7.5.
The appropriate fractions were combined to produce a
solid which was crystallized from water/acetonitrile and
then from water/isopropanol to give 267 mg. of L-alanyl-~-
chloro-D-alanine; m.p. 196-202C. (with decomposition);
[a]D + 1 (C, 1.0).
Examples 3_- 14
In like manner, the compounds shown in Table
I were made, identified by the melting point of the Na-Z-
dipeptide, and the m.p. and/or optical rotation (shown as
[~]D/concentration in lN HCl) of the dipeptide with the

113~
--4--
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1~374~8
5--
chemical formula of the compound. All degrees () are
in Centrigrade; "d" and "s" are used to show that the
compound decomposed or sintered at or before melting.
Where no optical identification is given for
the N-terminal aminoacid in the above table or in the fol-
lowing examples, a racemic mixture was used.
Example 15
In an ice bath, 1.07 g. of ~fluoro-D-alanine
in 20 ml. of methanol was treated with 1.1 ml. of SOC12.
The mixture was stirred two days at room temperature to
give a clear solution. Solvent evaporation and tritura-
tion with ether gave 1.28 g. of the methyl ester of
~F-D-Ala which melts at 130C. with previous sintering
above 110C.
A 630 mg. sample of this ester was treated
as in Example l(a), producing 738 mg. of amorphous Z-L-
Ala-3F-D-Ala-OMe.
A 620 mg. sample of the above N~ -protected
dipeptide ester was hydrogenated in the presence of 0.1 -
g. 5% Pd, 2 millimoles of hydrochloric acid and 100 ml.
of methanol. Evaporation of the solvent followed by
ether trituration and extensive drying gave an extemely
hygroscopic gum of L-Ala-~F-D-Ala-OMe~HCl; [~]D + 41
(c, 1.10).
In analogy to the above procedure, the cor-
responding ethyl or butyl esters are made by replacing
the above methanol with ethanol or butanol.
Example 16
a) A suspension of 512 mg. of the compound
of Example 1 in 10 ml. of DMF was stirred with 1.0 ml. of
acetic anhydride. After 90 minutes, the clear solution
was diluted with water, evaporated to dryness and the
residue ~laced on a chromatographic column containing
AG-l-X2/70Ac), (an anionic exchange resin sold by the
Dow Chemical Co.). Elution with 0.05 molar ammonium
acetate gave 475 mg. of the ammonium salt of N~-Ac-L-
Ala; [~]D ~ 21.2 (c, 1.3).
b) Acylation with N ~Z-L-Ala-ONSu followed by
hydrogenation as in Example 2 afforded the tripeptide
L-Ala-L-Ala-~F-D-Ala; m.p. 267-70C. (d).
~ ~r~d~lna~ k

46i~t
--6--
In the same fashion, Na-propionyl, Na-glycyl,
N~-valyl, Na-leucyl, and N ~ butyryl dipeptides are pre-
pared.
Example 17
In analogy with Example 1, the Na-carbo-
benzoxy derivative of L-a-aminobutyric acid was coupled
to ~F-D-Ala, followed by the usual deprotection reaction
to give L-aNH2-But-~F-D-Ala melting at 182C.(d). The
mentioned intermediate showed a m.p. of 169-70C.;
[a]D + 66 (c, 0.5).
Example 18
Using 3Cl-D-Ala in the procedure of Example
17 gave a Na-protected intermediate melting at 166-8C.
The dipeptide L-aNH2-But-~Cl-D~Ala melts at 171.5-2.5C.;
~a] + 17.1 (c, 0-5)-
Example 19
In a manner similar to Example l(a), Na-tert-
butyloxycarbonyl-L-norvaline was coupled to ~F-D-Ala to
yield the t-BOC-L-norvalyl-~F-D-Ala. It was then depro-
tected as follows:
A solution of 550 mg. of the above dipeptide
in 5 ml. tetrahydrofuran was added to 5 ml. 2N hydro-
chloric acid. It was evaporated under reduced pressure
to dryness after being stirred at room temperature for
17 hours. The residue was dissolved in 10 ml. ethanol.
2 m. of propylene oxide was added. After stirring in
cold room for 24 hours, it was filtered yielding 250 mg.
L-Norval-~F-D-Ala. Recrystallization from water gave
pure dipeptide; m.p. 207C (d); [a]D + 52 (c, 0.5).
Example 20
A suspension of 2.34 g. of D,L-a-amino-
octanoic acid in 30 ml. of water containing 2.52 g. of
NaHCO3 was stirred in an ice bath with a solution of
4.38 g. of carbobenzoxy-N-hydroxysuccinimidyl carbonate
in 30 ml. of 1,2-dimethoxyethane. After 3 hours, the
temperature was allowed to adjust to room temperature
and stirring was continued for three days. The resulting
solution was cooled in an ice bath and acidified with

11~'74~
c~ CI -7-
2~ u~ to produce 2.1 g. of the desired protected amino
acid; m.p. 89-92C.
The active N-hydroxysuccinimide ester of
the above was made in known fashion; it melts at 90-
5C. This material was coupled to ~F-D-Ala in the
fashion shown in the preceding examples. The N ~pro-
tected dipeptide melts at 116-22C., while the desired
D,L- ~amino-octanoyl-3F-D-Ala melts at 185-92C.;
[~ D + 19 (c, 0.5).
Other compounds of the above general descrip-
tion can easily be made by repeating Example l(a) but
using the succinimide esters of other N ~protected amino
acids. For instance, if said ester is that of isoleucine
or ~aminocaproic acid, the corresponding compounds are
obtained where R represents L-isoleucyl or L-~-amino-
aminocaproyl. Obviously, other amino acid esters carry-
ing protected additional functional groups can be em-
ployed to make the dipeptides of the current invention.
Particularly, the L-threonyl-, L-tryptophyl- and L-tyrosyl-
~-fluoro (or chloro)-D-alanines can be made by the above
route. In all cases, the functional groups, where
present, can be temporarily protected in known fashion
by benzyl, carbobenzyloxy, tert. butyl or othe protective
groups commonly used in the peptide art.
In order to show the pronounced synergistic
activity of the new compounds with D-cycloserine, reference
is made to the following in vitro tests.
In a two-fold agar dilution assay with E. coli
(Juhl) and E. coli 6880 as test organisms, compounds of
Examples 1 and 2 show a minimum inhibitory concentration
(M.I.C.) of~800 ppm. D-cycloserine alone shows a M.I.C.
of 12.5 ppm against the former and 6.2 ppm against the
latter _. coli strain. The combination of the new peptide
with D-cycloserine produces the following M.I.C. test re-
sults.

--8--
Co~bination E. coli (Juhl) E. coli 6880
Example Ratio M.I.C. M.I.C.
1:8 0.2:1.56 0.1:0.78
1 1:1 0.2:0.2 0.1:0.1
8:1 0.39:0-05 0-39:0-05
. . _
1:8 0.2:1.56 0.1:0.78
2 1:1 0.78:0.78 0.78:0.78
8:1 0.62:0.78 3.1:0.39
As shown, the compounds of the current inven-
tion allow the use of much lower concentrations of both
compounds to get the desired antibacterial results.
The in vitro activity of the halogenated pep-
tides and the halogenated peptide-antibiotic combinations
provided by the present invention can be demonstrated as
follows:
The halogenated peptides alone, the antibiotic
alone or mixtures of the halogenated peptides and selected
antibiotics are prepared in sterile concentrated aqueous
solutions at the desired ratios. Serial dilutions are
made to give a range of concentrations of the test sub-
stances. Samples of the dilutions are mixed with an ap-
propriate sterile synthetic medium in test tubes. The
tubes are then inoculated with an appropriate test orga-
nism and incubated at 35-37C. for 16-20 hours. Minimum
inhibitory concentrations, i.e., that concentration
which inhibits visible growth, are read and the frac-
tional inhibitory concentration indices (F.I.C.) are
calculated. The results obtained using representative
- halogenated peptides and representative antibiotics are
given in Table II. In all instances, E. coli (Juhl) was
used as the infecting microorganism.

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1:~3'~
--10--
The _ vivo activity of the halogenated peptides
and the halogenated peptide-antibiotic combinations pro-
vided by the present invention can be demonstrated as
follows:
Charles River mice, weighing approximately 20
g. each, are infected intraperitoneally with 10-100 times
the LD of the infecting organism. At predetermined inter-
vals post-infection, e.g., 1 and 5 hours, mice are dosed
subcutaneously with graded doses of the halogenated
peptide, antibiotic and combination thereof. The number
of mice surviving each treatment for 7 days post-infec-
tion is observed and the CD50 is calculated. The frac-
tional inhibitory concentration (F.I.C.) for each combi-
nation is calculated in the usual manner. The results
using D-cycloserine as an example of the antibiotic and
representative halogenated peptides are shown in Table
III.
In this table, the following infecting microor-
ganisms were used:
S. aureus (Smith)
_. coli (Juhl)
E. coli (305-101)
Strep. pyrogenes (C 203).
The infecting organisms are listed by the above code; the
compounds are listed by their Example number. In all in-
stances, the dipeptide and D-cycloserine were tested at a
ratio of 10:1. The CD50 combination column lists only
the amount of peptide present; the D-cycloserine amount
is 10% of the listed amount.

'74~i~
--11--
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._ .

-12-
The compounds of the present invention can be ad-
ministered intramuscularly, orally, subcutaneously or intra-
venously. Sterile, liquid dosage forms can easily be pre-
pared for parenteral administration by dissolving the above
dipeptide in the form of a water-soluble, non-toxic salt in
isotonic sodium chloride solutions containing optional buffers,
stabilizers, and/or preservatives. Liquid oral dosage forms
in the form of elixirs, syrups or suspensions can be made
in standard fashion, also optionally containing the above
additives together with coloring or flavoring agents.
Solid dosage forms for oral administration include
tablets, capsules, pills and wafers. For these dosage forms,
the usual solid diluents are used where required. Capsules
can be filled with undiluted powdered or granulated crystals
of the new compounds. For tablets, the following standard
procedure may be used:
About one-half of 50 g. of cornstarch is milled
together with 50 g. of the above dipeptide and 220 g. of
calcium phosphate dibasic dihydrate. This blend is milled
until homogenous and passed through a 40-mesh screen. The
remaining portion of the cornstarch is granulated with water,
heated and mixed with the above drug blend in a hot air
oven at 50C and sifted through a 16-mesh screen. These
granules are then mixed with 16 g. of talcum powder, 4 g. of
magnesium stearate and 0.8 g. of combined coloring and fla-
voring additives. The mixture is blended to homogeneity,
passed through a 30-mesh screen and blended for another
15 minutes. This blend is compressed into tablets weighing
approximately 350 mg. using a 9/32" standard convex punch
resulting in tablets of a hardness of 7-9 with each tablet
containing 50 mg. of the drug. In a similar fashion, tab-
lets weighing 600 mg. containing 250 mg. of drug can be
prepared, preferably in a tableting machine producing bi-
sected tablets.
While the above examples are directed to the
peptides per se, the acid addition salts can readily be
prepared in known fashion. The nontoxic salts useful as

-13-
antibacterials include primarily the hydrochloride, phos-
phate, sulfate, acetate, succinate and citrate.
As will be seen from the above examples, the
current dipeptides are antibacterially active in warm-
S blooded animals. Against certain bacteria, the new di-
peptides are powerful synergists for known antibacterials,
enabling the use of the latter in quantities of only a
small fraction of its curative dose. In particular,
by combining the current dipeptide with a medicinally
useful antibiotic in a weight ratio of 1:1 to 10:1,
excellent antibacterial synergism is observed. While
the demonstrated synergistic results above are based
on the use of specific antibiotics, it will be under-
stood that other antibiotics including penicillins other
than the above carbenicillin, cephalosporins other than
cephalothin, streptomycin, erythromycin, tetracyclin,
etc. can be combined with the new peptides to obtain
better results than with such antibiotics alone.